DE2547557A1 - MEANS AND METHOD OF INTERRUPTING ENZYME REACTION - Google Patents

Description

Priority: October 23, 1974, Japan, No. 122 243

Means and method for interrupting an enzyme reaction

The invention relates to a gown for interrupting a reaction, which is suitable for measuring the activity of serural enzymes, which are used as a diagnostic feature for the diagnosis of liver, heart and Muscle disorders in humans are used.

The invention relates particularly, the new application of a wäesr.ig alkaline solution of Cyclohexylaininopropansulfonsäure with a specified range of concentration and pH-V ^»r erts as interrupting means for interrupting the enzymatic reaction of dehydrogenase or a dehydrogenase system in the spektrophotoraetrischen method for measuring the activity of serum enzyme in the above-mentioned enzyme or enzyme system.

The invention therefore relates to a method for interrupting the enzymatic reaction of dehydrogenase or a dehydrogenase system in the spectrophotometric Kessung of the activity of the serum enzyme in the specified enzyme or enzyme system, which is characterized in that an aqueous alkaline Solution of cyclohexylaminopropanesulfonic acid sit with a concentration of 0.1 to 2 M and a pH of 10.5 to 12.5 is used.

On suitable spectrophotometric measuring methods for measuring the

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Activity of serum enzyme in a dyhydrogenase or dehydrogenase system which can be used according to the invention include various measurement methods commonly used in this field for the stated purpose. Appropriate Examples thereof are the measurement of ultraviolet absorption (hereinafter referred to as "ultraviolet measurement") in which the activity of glutamic acid-oxaloacetic acid transaminase (hereinafter referred to as GOT), glutamic acid pyruvate transaminase (hereinafter referred to as GPT), aldolase (hereinafter referred to as ALD), creatine phosphokinase (hereinafter referred to as CPK), Lactic acid dehydrogenase (hereinafter referred to as LDH), alcohol dehydrogenase or sorbitol dehydrogenase in human serum by measuring the change in the absorption of nicotinamide adenine dinucleotide (hereinafter referred to as NAD) or nicotinamide adenine dinucleotide phosphate (hereinafter referred to as FADP) is determined at 340 nm, which is the reaction medium by itself or in combination with a suitable dehydrogenase or a dehydrogenase system. It also includes the fluorescence emitting method (hereinafter referred to as "fluorescence method") in which the serum enzyme activity of the above specified enzymes in human serum by alteration. the intensity of the fluorescence of FAD or NADP upon excitation with a radiation of 340 nm is measured, with FAD or FADP the reaction medium either alone or in combination. with a dehydrogenase, as well as similar methods.

The invention wix ^ d explained in more detail below, wherein from For the sake of convenience, reference is only made to the ultraviolet measurement method. However, it can be seen that the interruption means according to the invention in a more satisfactory manner Way can also be applied to any other measurement method, such as the fluorescence measurement method and the like.

An increase in GOT or GPT activity in serum occurs in some liver disease or heart attack, so measuring this activity is a useful means of diagnosing this Diseases is considered.

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2 b 4 7 b b 7

To measure serum GOT or GPT activity, the reaction can be carried out, but maleate dehydrogenase in the case of GOT and LDH in the case of GPT along with the enzyme substrates such as <? 0-ketoglutaric acid and L-aspartic acid for the former Enzyme or cc-ketoglutaric acid and I-alanine for the latter Enzyme and a reduced form of NAD (hereinafter referred to as NADH) is added to the reaction system in either case. the end Aspartic acid is formed with GOT oxaloacetic acid (when using GPT, pyruvic acid is formed from alanine and the When using GPT obtained products or applied enzymes are mentioned below in brackets). This oxaloacetic acid (Pyruvic acid) is converted into malic acid (lactic acid) with the help of maleate dehydrogenase (lactic acid dehydrogenase) and at the same time, NADH is oxidized to NAD, with a decrease in the absorption caused by NADH takes place at 340 nm. The amount of oxaloacetic acid (pyruvic acid), which is formed by GOT (GPT) is stoichiometrically equal to the amount of oxidized NADH and the activity of GOT (GPT) can therefore be determined by the amount of oxidized NADH obtained from the decrease in absorbance at 340 nm.

Serum ALD activity becomes more progressive in liver disease Muscular dystrophy or heart attack increased. To measure the ALD activity, fructose-1,6-diphosphate can be added to the reaction medium as a substrate together with triose phosphate isomerase (hereinafter referred to as TMI) and glycerol-1-phosphate dehydrogenase (hereinafter referred to as GDH) may be added. In this system, the compounds through ALD are converted into dioxyacetone phosphoric acid which is then converted into glycerol-1-phosphate under the action of GDH and NADH. At the same time, NADH oxidized to NAD. The hydrolysis of 1 mole of fructose-1,6-diphosphate with ALD leads to the oxidation of 2 moles of NADH.

CPK is found in serum in case of skeletal muscle damage or Heart muscle increased. Creatine phosphoric acid is used to determine CPK activity and adenosine 5'-diphosphate (hereinafter referred to as ADP) are used as substrates and added to the reaction medium also hexokinase (hereinafter referred to as HK), glucose, GIu-

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cose-6-phosphate dehydrogenase (hereinafter referred to as G6PDH) and NADP added. Through the action of CPK, ADP is converted into adenosine 5'-triphosphate (hereinafter referred to as ATP), while the glucose in the reaction medium is converted into glucose-6-phosphate under the action of ATP and HK. Thereafter the glucose-6-phosphate is converted into 6-phosphogluconic acid under the action of G6PDH and NADP and at the same time with this conversion, NADP is converted into its reduced form, hereinafter referred to as NADPH, with the absorption increases at 34Q nm. The amount of ATP that is formed by CPK is stoichiometrically equal to the amount of reduced NADP.

In the case of serural enzymes other than those listed above, is a similar reaction system is applicable. When the serum enzyme itself is a dehydrogenase, like LDH, so the 'enzyme activity is due direct dehydrogenation of NADH or NADPH with a substrate is determined, whereby the absorption intensity is changed at 340 nm.

As stated above, a characteristic of the ultraviolet measurement method is that the measurement can be carried out using a dehydrogenase reaction • in order to reduce the reaction of the above-mentioned serum enzyme in to be coupled in each case with the reaction of the oxidation of NADH (or NADPH) or the reaction of the reduction of NAD (or NADP).

The ultraviolet measurement method does not require any staining procedures and can be carried out with the aid of extremely simple procedural steps in the determination, in contrast to others Measurement methods in which, after the coloration of the reaction product or the remaining substrate, a colorimetric determination must be carried out.

The absorption intensity of NADH or NADPH at 340 nm is also fully proportional to the concentration of these compounds and the molar extension coefficient is sufficiently high. With With the help of the ultraviolet method, remarkably high precision, accuracy and reproducibility can be achieved compared to other achieve their known methods. The indication of an activity

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value (unit of activity), which has been recently identified by the International Union of Biochemistry can easily be done by changing the absorbance at 340 nm is multiplied by a coefficient. In view of this additional benefit mentioned, it was finally assumed that the method of ultraviolet measurement, which was previously used in basic biochemical research, to carry out of routine determinations of serum enzymes in clinical diagnostic laboratories is applicable «

The previously used methods of ultraviolet measurement, which are carried out manually, are not widely used, because in the absence of an interrupting means suitable for the above-mentioned enzyme reaction it is difficult to analyze large numbers of serum samples. In addition, in cases where numerous serum samples are analyzed daily, as is the case in a clinical diagnostic laboratory, a special photometer can be used which is specially designed for the Method of ultraviolet measurement has been developed, such as an IKB-8600 reaction rate analyzer (LKB Co., Sweden), a Rotoehem II-Fast Analyzer (Aminco Co., USA), etc. Specifically, in manual methods using a conventional photometer to perform ultraviolet measurement a reaction mixture and a serum sample are placed in a cell and the change in absorbance at 340 nm in Measured over a period of 10 to 30 minutes as a function of time. Because This time-consuming method limits the number of serum samples to be tested per day to a maximum of 10 to 20 and the Carrying out these measurements for a whole day is very tiring for the analyst. In addition, this is the one used so far specific photometer a very expensive device and requires a high capital investment, so they are for medium-sized and small clinical diagnostic laboratories are not suitable.

A considerable number of interrupting means for common enzymatic reactions have been proposed in the art, which, however, are not applicable to the ultraviolet method for the following reasons. Deproteinizers, such as tri-

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chloroacetic acid, perchloric acid, metaphosphoric acid, sulfuric acid-tungstic acid and the like reduce the "absorption of I ¹ JADH (or NADPH) at 340 nm, and if other acids are added to the reaction mixture in an amount sufficient to stop the reaction, the absorption will also be decreased at 340 η. On the other hand, when an aqueous solution of an alkaline agent such as sodium hydroxide is added in an amount sufficient to stop the reaction, the absorption of NAD (or NADH) at 340 nm undergoes irregular changes. When the enzyme reaction is stopped by heat treatment is, the reaction mixture becomes cloudy due to the serum protein and by conventional centrifugation at 3000 rpm or less the cloudiness can only be partially removed and no clear solution suitable for measurement can be obtained.

It has also been reported in the prior art that an aqueous alkaline solution of p-chloro mercuribenzoate was used as a disconnecting agent can be used for the dehydrogenase reaction (E. Raabo, Scandinavian Journal of Clinical & Laboratory Investigation, 17, - (1965), ■ 265). However, it was confirmed that this solution only leads to an inhibition of the dehydrogenase reaction • if there is a narrowly limited buffer concentration in the reaction mixture prevails and that in addition only a partial inhibition of the dehydrogenase reaction is achieved if a reaction. a mixture of commercially available diagnostic agents is used / ended. This known solution is therefore not suitable for the practical one Use.

The invention is therefore based on the object of satisfying this demand for suitable interruption means. With far-reaching Investigations to solve this problem have been found that numerous different dehydrogenases completely can be inhibited if an appropriate amount of an aqueous alkaline solution having a specific concentration is used on cyclohexylarainopropanesulfonic acid (hereinafter referred to as GAPS) and has a specific pH and shows strong buffering action within an alkaline pH range. These Solution does not cause any change in the absorption of NAD or

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⁷ '2 b 4 V 5 b 7

NADP in the area of 340 µm and also shows ideal characteristics as interrupting means without in any way the Turbidity of the serum of normal healthy patients as well as of lipemic serum affected.

It is also an object of the invention to provide a new method for interrupting the enzymatic reaction of a dehydrogenase or a dehydrogenase system in the spectrophotometric measurement method to provide. This problem is solved by using an aqueous alkaline solution of CAPS.

Other objects and advantages of the invention are from the following Description visible.

By using the interrupting means according to the invention, it becomes possible to carry out the reaction in numerous serum samples at the same time perform, then interrupt the reaction by adding the interrupting agent after a certain period and then the enzyme activity by measuring the absorbance at 340 nin to determine. With the help of this procedure it becomes possible for a single person to do the respective activity on 100 to 200 Can determine serum samples per day.

In the pail in which the activity over time in a Cell is measured, it is impossible to reduce the volume of the reaction mixture to a certain value because a a certain capacity of the cell is required. In contrast, it is when applying a method using the inventive method Interrupting means possible that the amount of the reaction mixture to be converted is reduced by the amount of to be added interrupting means is set, since it is sufficient if the final volume after the addition of the interrupting means the required limit of the capacity for the Measurement exceeds.

In general, it is easy to reduce the volume of the reaction mixture to a value as low as 1/3 to 1/6 that of the usual manual method to decrease. A blank sample for the serum can be tested without adding the reaction mixture and · a blank sample from the reagent is measured separately. Of the

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The blank value for the experiment is given as the sum of the above determined for both blank values. So no reagent is consumed for the correction by the blank sample, thus making the necessary Savings on reagent can be obtained in clinical laboratories.

More detailed details of the invention and of the invention Procedures are explained in more detail below.

Effect_of_ an_alkaline_puff ££ solution_on_the Dehydrogenase_activity_and_influence_on_absorption_inks

As a result of the investigations on which the invention is based on phosphate buffer solution, glycin buffer solution, arginine buffer solution, Guanidine buffer solution and CAPS buffer solution have been found to have some disadvantages, buffer solutions other than CAPS buffer solution and are not suitable to be used as a means of interruption.

, Phosphate buffer solution has the effect of reducing auto-oxidation noticeably promoted by NADH. This effect can be prevented by adding a suitable amount of ethylenediaminetetraacetic acid (ÄDTE) be, but with the absorption of NAD at 340 "nm by adding a phosphate buffer solution containing ÄDTE to the enzyme reaction mixture which contains an amino acid, for example glycine, is greatly increased, thus causing a non-enzymatic change in NAD. A corresponding change the absorption can be caused if a glycine buffer solution or an arginine buffer solution to a reaction mixture containing ADTE is added. This causes an increase in the absorption of NAD at 340 nm. A guanidine buffer solution does not show this effect, but the solution itself may reduce the absorption of NAD at the concentration and pH required for the Inhibiting the action of an enzyme necessary to increase it. This effect of increasing the absorption of NAD may over time fluctuate constantly and it is therefore difficult to

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to correct gentle influence.

(2) Inhibitory effect_of_CAPS buffer solution_on_the_drogenase reaction

Four enzymes that are widely used for testing "in clinical Diagnosis among the different serum enzymes applied were in terms of the inhibitory exerted by CAPS buffer solution Effect tested. Some of these attempts are summarized in Table 1. In these experiments the changes were made the absorbance at 340 nm during the entire process of the reaction automatically recorded and expressed as the degree of inhibition. To carry out the reaction, serum becomes a Reaction mixture given and then the CAPS buffer solution in different concentrations at different pH values and added in different amounts.

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a b e 1 1

CAPS buffer solution concentration
(m) 3) pH 3) Concentration for the full
constant inhibition with water
niger NaOH solution (Endlcon-
centering) Serum enzyme 1) GOT GPT AID CPK Added
lot 0.5 12.0
12.5 C 4)
C. C.
C. C.
C. C.
C. 1/2 vol. 1 11.0
11.5 80
C. C.
C. C.
G C.
C. 1 full 2 10.5
11.0 65
C. C.
C. C.
C. C.
C. 2 vol. . 0.5 11.0
11.5 85
C. C.
C. C.
G C.
C. 4- vol. 1 10.5
11.0 70
C. C.
C. C.
G C.
C. 2 10.5 C. C. C. c ■ 0.1 11.0
11.5 10
35 70
C. 95
C. 95
C. 0.125 11.5 80 C. C. C. 0.5 10.5
11.0 85
C. C.
C. C.
C. G
C. 0.1 11.5 C. C. C. C. 0.125 11.0 C. C. C. C. > 0.15n > 0.15n > O, ln > O, ln

1) Diagnostic reagents from Calbiochem USA, for the determination of GOT, GPT and CPK, and diagnostic reagents from Boeringer Mannheim, for the determination of AID, human standard serum (Cal-trol abnormal) the Calbioehem, for use in serum.

2) Volume ratio of the added buffer solution to the enzyme reaction mixture. The designation 1/2 volume means for example

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wise that half a unit volume of buffer solution per unit volume of the reaction mixture is added.

3) Molar concentration and pH of the added buffer solution.

4) C means 100% complete inhibition and a corresponding one Number means the percentage of partial inhibition.

a) Influence on the absorption intensity

By adding a CAPS buffer solution to a coenzyme solution usually the absorption intensity of the coenzyme at 340 nm increased to some extent, although depending on the concentration and the pH of the buffer solution larger or smaller differences can be observed. Increasing the absorption of the however, the oxidized form and the reduced form of the coenzyme are approximately the same per u-mole unit and, if so, working curves by measuring the absorbance at 340 nm at various ratios of the oxidized form to the reduced form, held constant of the total amount of coenzyme are taken up, a curve is obtained when CAPS buffer solution is added, the Slope is equal to the curve obtained without addition of the buffer and that obtained by moving that without the buffer solution recorded curve in the direction of increased absorption intensity can be obtained.

With the ultraviolet measuring method, the enzyme activity is determined by the The difference in absorbance at 340 nm before and after performing the reaction is determined, and therefore the increase in absorbance is determined switched off by the CAPS buffer solution.

b) Influence on the stability of the absorption intensity

As explained above, it was found that the absorption of the coenzyme at 340 nm after the dissolution is increased to a certain extent by the addition of CAPS buffer solution according to the invention. Some the tests on the stability of the absorption after the solution are summarized in Table 2. In this table the Change in the absorption intensity at 340 nm over 3 hours

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shown after dissolving the coenzyme in CAPS buffer solution,

Table 2

3APS ¹ ^ -buffer-
solution NAD NADH NADP NADPH 3.5 m; pH 11.5 Ο, ΟΙΟΑ / h / cra ² ^
Not a bigger one
change
the OD Uncompromised
changes Uncompromised
changes Unchanged
changes 3.33 m; pH 11.5 0.005A / h / cm
Not a bigger one
change
the OD ti Il It 3.25 m; pH 11.5 τ)
Unchanged ' ti It It 3.25 m; pH 11.5
(containing
L2.5 mmol ADTE) It H It It 3.25 m; pH 12.0 0.005A / h / cm
Not a bigger one
change
the OD Il Il dd 3.25 m; pH 12.5 0.010A / h / cH
leash bigger
change
the OD It 0.005A / h / cm
Not a bigger one
change
the OD It

1) The coenzyme is in the specified buffer solution in a Concentration of 1.1 χ 10 "~ ^ m dissolved.

2) Change in the absorption per hour when measured with the aid of a cell with an optical path length of 1 cm.

3) Change in absorption of ί 0.002A or less during Hours immediately after dissolution are said to be "unchanged".

As can be seen from Table 2, all coenzymes are in buffer solutions

at a concentration of 0.25 M or less and a pH of 11.5 or less, and are stable even when ADTE is at a concentration of 12.5 mmol or less. on the other hand the stability of NAD is somewhat reduced when the concentration is 0.33 M or more or the pH is 12 or more. When the pH is 12.5 or more, the stability of NADP is also decreased, while the stability is decreased the reduced form of the coenzyme is not affected.

When NAD or NADP in an aqueous solution of a strongly alkaline Gowns, e.g. B. sodium hydroxide or potassium hydroxide is dissolved, the absorption intensity increases at 340 nm over time to a maximum, which is reached after 10 to 20 minutes, and then gradually decreases again. These Changes occur continuously for several hours and the higher the alkali concentration, the greater the observed Change * It is therefore extremely difficult to find conditions among which the enzyme reaction is interrupted without affecting the absorption of the coenzyme if an aqueous one Solution of a strong alkali without a buffer effect is used.

. (4) Single luß_auf_die _g _Trübung _<> _des_Serums

In determining enzyme activity, the pre-reaction absorbance is usually measured by adding serum to the reaction mixture and CAPS buffer solution is added immediately to interrupt the reaction. By simplifying the above The following advantageous method can be used, since the addition of the CAPS buffer solution reduces the turbidity of the Serum not affected.

The absorption of the reaction medium containing CAPS buffer solution is predetermined and then this is the absorption of the serum added at 340 nm, v / o is determined by the absorbance before the reaction. This method can save on reagent since no reagent is used to determine the absorbance of each serum prior to the reaction.

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When carrying out the method according to the invention should.die Concentration of CAPS in the aqueous alkaline solution within a range from 0.1 to 2 molar, preferably 0.1 to 0.5 molar and in particular 0.5 molar. The pH of the aqueous alkaline solution of CAPS should be within a range from 10.5 to 12.5, preferably 11.5 to 12.5 and in particular 11.5. As alkalis, for the purposes of the invention can be used, for example, alkali metal hydroxides, such as potassium hydroxide, sodium hydroxide, lithium hydroxide or ammonium hydroxide and the like. It should be noted that alkaline substances other than those mentioned above can also be used are used, provided that they do not interfere with the conditions of the analysis, the results of the analysis and the like.

It is also desirable to add a water-miscible aliphatic alcohol to the aqueous alkaline solution of CAPS. Suitable examples of alcohols suitable according to the invention are methanol, ethanol, n-propanol, isopropanol and the like. The amount of the alcohol to be added is not an essential feature, but usually and preferably the alcohol is used in an amount of 10 to 30 fo (Y / V). In general, methanol or ethanol can practically be used in an amount of 30 % . n-Propanol can be used in an amount of 10 % and isopropanol in an amount of 20%.

The invention will now be further elucidated by the following examples explained.

example 1

Measurement; of GOT activity in Ser

(a) Two test tubes A and B were used for a serum sample. 1.0 ml of a pH 7.4 reaction mixture containing the following ingredients was added to each test tube indicated amounts contained.

The enzyme preparation in units (international Units) and the other components are given in µmoles.

80 98 1 87 1102

25475 57 100 7.5 36 16 0.8 0.17 0.33 unit 0.33 unit

Tris (hydroxymethyl) aminomethane
ex, -ketoglutaric acid
L-aspartic acid
Succinic acid
Sodium bicarbonate

Reduced form of nicotinamide adenine dinucleotide

Malic acid dehydrogenase
Lactic acid dehydrogenase

The test tubes were incubated in a water incubator at 30 ° C. and after 5 minutes, 25 μl of serum were added to one test tube A and mixed well. After the subsequent incubation for 60 minutes, 1.0 ml of a 0.5 M CAPS buffer solution (pH 11.5) was added to each of the two test tubes A and B, and then 25 μl of serum were added to test tube B. Both test tubes were shaken thoroughly. 3 hours after the addition of the CAPS buffer solution, the absorption of the solution in the
Test tubes A and B measured at 340 nm using a cell with an optical path length of 1 cm.

The GOT activity was calculated according to the following equation.

The absorbance of the solution in test tube A is called A and the im
Test tube B designated as B:

(B-A) χ 217 = Milliunits (international unit) / ml

serum

(b) Using the same procedure as in the experiment described above under (a), but with the modification that instead of of the 0.5 M CAPS buffer solution a 0.2 M CAPS-30 # (V / V) -methanol buffer solution (pH 11.5) was used, the same results were obtained.

6 0 9 8 18/1 1 Π?

Example 2 Measurement; the G-PT activity in serum

(a) In this experiment, two test tubes A and B were used for a serum sample. A 1.0 ml portion of a reaction solution of pH 7 _f 4 having the composition shown below was poured into each test tube. The units of the fourth given are the same as defined in Example 1 (a).

Tris ~ (hydroxymethyl) aminomethane £ 100> 0-ketoglutaric acid 8.17

! -Alanine 16.5

Succinic acid. 35 sodium hydrogen carbonate 0.8

Reduced form of nicotinamide

adenine dinucleotide 0.17

Lactic acid dehydrogenase 0.33 unit

The test tubes were incubated in a water incubator at 30 ^° C. and the subsequent process steps were carried out in the same way as in example 1 (a). GPT activity was calculated using the equation given in Example 1 (a).

(b) Using the same procedure as above under (a), but with the modification that instead of the 0.5 m CAPS buffer solution a 0.2 m CAPS-20 $ (V / V) isopropanol buffer solution at pH 11.5 was used, the same results were obtained.

Example 3 Measurement of AIP activity in serum

(a) Two test tubes, A and B, were used for a serum sample A 1 ml portion of a reactant solution was placed in each test tube of pH 7.4, which contained the ingredients indicated below. The units of the values given were

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same as defined in Example 1 (a).

Collidine buffer 56 monoiodoacetic acid 0.3

Fructose-ljö-diphosphoric acid 3

Reduced form of nicotinaiaid adenine dinucleotide 0.17

Glycerol-1-phosphoric acid dehydrogen

nose 0.35 unit

Triose phosphate isomerase 2 units

The test tubes v / ere in an incubator at 30 ⁰ C and incubated 5 minutes v / ere 30 pl serum in the test tube A was added and mixed in well. After incubation for 60 minutes each 1 ml was given a 0.5 m CAPS buffer solution of pH 11.5 in the test tubes A and B and then the serum in the test tube B were given 30 ul. Both test tubes were shaken thoroughly and the absorbance at 340 nm was measured.

The corresponding absorption values are designated as A and B. and the activity is calculated according to the following equation: (B-A) χ 90.6 = milliunits / ml serum

(b) Using the same procedure as under (a), but with the modification that instead of the 0.5 m CAPS buffer solution one from 0.2 ra CAPS and 10 # (V / V) n-propanol existing buffer solution are used the same results were obtained.

Example 4 Measurement of CPK activity in serum

Two test tubes were used for a serum sample and placed in the 1 ml of a reaction solution having a pH of 7.4 and the composition shown below was poured into individual test tubes. the Units for the given values are the same as in

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Example 1 (a) were defined.

Sodium salt of F, N ^f -bis- (2-ethanosulfonic acid) -piperazine 50.

Sodium carbonate 20

Disodium creatine phosphate 20 Trilithium adenosine 5 '- diphosphate 1.5

Glucose 15

Magnesium asparaginate 10

Adenosine ^ -5'-phosphoric acid 10 Glutathione 8.7

letra sodium ethylenediainine tetraacetate 1.66

Oxidized form of nicotinamide adenine dinucleotide phosphate 0.6

Hexokinase 1 unit

Glucose-δ-phosphoric acid dehydrogenase 0.33 unit

The test tubes were placed in a v / water incubator at 30 ° C for 5 minutes incubated and then 50 μl of serum was placed in each of the test tubes A and B given and mixed in. The incubation was continued and after 15 minutes and 60 minutes each 2 ml of a 0.1 m CAPS-5mMol ÄBTE buffer solution (pH 11.5) into test tubes A and B given to interrupt the reaction. The time after the addition of the CAPS buffer solution, at which the absorbance at 340 nm is measured, became the same for both test tubes A and B Value set.

The absorption of the liquids in test tubes A and B. is designated as A or B and the activity is calculated using the following formula:

(A-B) χ 218 = milliunits / ml serum

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Claims (17)

Sponsorship claims 1. Method for interrupting the enzymatic reaction of a dehydrogenase or a dehydrogenase system when carried out the spectrophotometric analysis to determine the activity of a serum enzyme in the specified enzyme or enzyme system, thereby characterized in that the means for interrupting the enzymatic reaction is an aqueous alkaline solution of cyclohexylaminopropanesulfonic acid at a concentration of 0.1 up to 2 m, which has a pH of 10.5 to 12.5. 2. The method according to claim 1, characterized in that that one carries out the interruption of the enzymatic reaction in the spectrophotometric analysis in which the change the absorption of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate is measured at 340 nm. 3. The method according to claim 1, characterized in that the interruption of the enzymatic reaction at performs the fluorescence analysis, in which the change in the fluorescence intensity of nicotinamide adenine dinucleotide or Nicotinamide adenine dinucleotide phosphate when excited with light a A wavelength of 340 nm is measured. 4. The method according to any one of claims 1 to 3, characterized in that one uses an interruption means, that in the aqueous alkaline solution also has a water-miscible one Contains aliphatic alcohol. 609 «1R / 1 in? 5. The method according to any one of claims 1 to 4, characterized in that the interruption means in one Concentration, from 0.1 to 0.5 m. And with a pH of 11.5 to 12.5 used. 6. The method according to any one of claims 1 to 4, characterized in that the interrupting agent is used in a concentration of 0.5 m and with a pH of 11.5. 7. The method according to claim 4, characterized in that that the water-miscible aliphatic alcohol used is methanol, ethanol, n-propanol or isopropanol. 8. The method according to claim 4, characterized in that the alcohol is used in a concentration of 10 to 30 % . 9. The method according to claim 7, characterized in that that isopropanol is used in a concentration of 20 ^. 10. The method according to any one of claims 1 to 4, characterized in that one uses an interruption means, its concentration is 0.2 m and its pH value is 11.5, which contains isopropanol in a concentration of $ 20. 11. The method according to any one of claims 1 to 4, characterized in that 609818/110? indicates that the alcohol used is methanol or ethanol in a concentration of 30 % . 12. The method according to any one of claims 1 to 4, characterized in that the alcohol used is n-propanol in a concentration of 10 % . 13. Means for interrupting the enzymatic reaction of a dehydrogenase or a dehydrogenase system in the spectrophotometric Analysis for determining the activity of a serum enzyme in the enzyme or enzyme systems according to any one of the claims to 12, characterized in that it is made of active Substance an aqueous alkaline solution of cyclohexylaminopropanesulfonic acid with a concentration of 0.1 to 2 m with a pH value from 10.5 to 12.5. 14. Means according to claim 13 »characterized in that that the aqueous alkaline solution also contains a water-miscible aliphatic alcohol. 15. Means according to claim 14, characterized in that that it contains methanol, ethanol, n-propanol or isopropanol as alcohol. 16. Composition according to claim 15, characterized in that it contains isopropanol as alcohol in a concentration of 20%. 6 0 9 8 18/110? 17. Composition according to one of claims 13 "to 16, characterized in that it has a concentration of 0.2 m, a pH value of 11.5 and contains isopropanol in a concentration of 20 io . 609818/1 102